This invention relates to the field of cellular biology and disease states, including HIV infection and diabetes. Specifically, this invention provides novel assays for the effects of antiretroviral protease inhibitors on the Glut4 glucose transporter and a novel in vivo model of insulin-resistant glucose transport disease states.
Several publications are referenced in this application in order to more fully describe the state of the art to which this invention pertains. Each of these publications is incorporated by reference herein.
The development of new targets for therapeutic agents for the treatment of HIV infections, as well as powerful combinations of those therapeutic agents has led to what is now commonly referred to as HAART (for highly-active antiretroviral therapies). Among the cornerstones of the therapeutic components of these HAART approaches are the antiretroviral protease inhibitors which have been developed. As part of combination therapies, HIV protease inhibitors play a critical role in suppressing viral titers and increasing CD4+ lymphocyte counts, which can result in significantly reduced mortality among HIV patients.
The human immunodeficiency virus (HIV) genome encodes an aspartyl protease that is required to process its viral precursor polyproteins to their mature forms. This protease activity is essential for the proper formation of infectious HIV virions. The development of a class of specific agents that target the HIV protease was an extraordinary advance in the treatment of HIV infection.
Despite the rapid progress in the treatment of the disease, and the improved prognosis for those infected, it now appears clear that the use of protease inhibitors is associated with potentially serious side effects. In 1997, the FDA issued a Public Health Advisory to healthcare professionals warning that use of protease inhibitors was associated with increases in blood sugar and diabetes. Eighty-three cases had been reported, of which twenty seven required hospitalization. Diabetes cases were associated with use of several available protease inhibitors, including indinavir, nelfinavir, ritonavir, and saquinavir.
Although the mechanisms have not been elucidated, it is clear that protease inhibitor use is linked in some manner to a syndrome of alarming metabolic abnormalities characterized by hypertriglyceridemia, hypercholesterolemia, peripheral fat wasting, central adiposity and hyperglycemia and insulin resistance. The etiology of this metabolic syndrome associated with protease inhibitor use currently remains unknown, but its features are similar to those present in the insulin-resistant state commonly referred to as Syndrome X. Common symptoms of the syndrome include body fat changes including: enlarged dorsicervical fat pads (known as xe2x80x9cbuffalo humpxe2x80x9d); abundance of benign lipomas; deposition/accumulation of fat in the abdomen or viscera (Crix belly); breast hypertrophy; and a characteristic loss of fatty tissue from the face and extremities. Collectively these body fat changes are referred to as lipoatrophy, or more commonly, lipodystrophy. Hyperlipidemia and insulin resistance appear to occur with high prevalence among patients using protease inhibitors, such that increased risk of premature cardiovascular disease and diabetes are relevant issues. The prevalence of lipodystrophy has been reported to be as high as 83% according to one study. Other studies suggested that some of the symptoms may appear to varying degrees in patients not treated with protease inhibitors, or that symptoms vary depending upon which protease inhibitor was used for treatment. Yet other studies revealed possible differences in patients based on age, gender, length of infection and other factors such as change in weight and hemophilia.
Thus, sustained treatment with the currently available antiretroviral protease inhibitors results in at least some, or all, of these metabolic disturbances, particularly those associated with hyperlipidemia and insulin resistance. The long-term, or even near-term, risk to the health of patients, while less than the imminent risk of uncontrolled viral growth, is substantial. The ability to design drugs lacking the undesirable side-effects is widely recognized as a need in the art and would be of great significance in combating HIV and other retroviral diseases. Furthermore, a more detailed understanding of the molecular mechanisms which lead to this metabolic disorder would contribute to the development of novel experimental or in vivo models related to the generalized problem of insulin resistance.
It has now been discovered in accordance with the present invention that anti-HIV drugs of the protease inhibitor class, including, for example, indinavir, ritonavir and amprenavir, are selective inhibitors of the Glut4 glucose transporter. This discovery enables the development of assays for use in screening of protease inhibitors for this unwanted and undesirable side-effect; in addition, improved procedures for the rational design and testing of antiretroviral protease inhibitors are enabled by this novel method.
The present invention provides novel assays for determining the effect of protease inhibitors on glucose transport activity. The invention also provides methods for screening antiretroviral protease inhibitor drugs for side-effects related to glucose transport in the presence or absence of insulin stimulation. In its most basic and general form, the assay method comprises the steps of; providing glucose transporters of interest in a membrane system; incubating the glucose transporters with a measurable form of glucose or a glucose analog; adding to the incubation mixture a antiretroviral protease inhibitor being screened; optionally, stimulating the glucose uptake of the cells with insulin; and quantifying the glucose transport by determining the difference in the amount of glucose uptake in the presence and in the absence of the protease inhibitor being screened, and optionally, in the presence or absence of insulin, or other glucose transport-altering substances. The invention provides, in one embodiment, that the glucose transporter is a Glut4 isoform. In one basic embodiment the membrane system comprises cells, for example adipocytes, producing a glucose transporter. The invention provides a wide variety of cells for use with the methods.
Also provided in accordance with the present invention is a method for testing a wide variety of antiretroviral protease inhibitors for side-effects on glucose transport. Included among the protease inhibitors and their derivatives to be tested by the method above are various compounds and families of compounds, many of which are already established as inhibitors of aspartyl proteinases.
The invention also provides for a variety of glucose compounds to be used as the detectable glucose. Glucose compounds include glucose, and its analogs, including transportable analogs of glucose.
The invention also provides methods for measuring the side-effects of protease inhibitors on glucose transport in cell-free systems. The advantages of cell-free systems are well known in the art. In the cell-free methods of the invention, the membrane system selected comprises a naturally-derived membrane from cells in certain embodiments, while in others, a noncellular system, for example, artificial membranes or vesicles, is used with the glucose transporter isoforms.
Also provided for use in the methods of the present invention when the membrane system selected is cells, are cells containing one or more expressible nucleic acids encoding one or more glucose transporters. The invention provides that the cellular expression of the glucose transporter is a stable characteristic of the cell line in certain embodiments. In other embodiments, the expression of the glucose transporter is transient. The expressible nucleic acids encode one or more homologous or heterologous glucose transporters of interest. In one embodiment, the cells have a substantially negligible basal level of glucose transport, other than the glucose transport provided by the heterologous glucose transporter.
Also provided in accordance with the present invention are kits for testing protease inhibitors for side-effects involving glucose transport. The utility of such kits is well established. The kits of this invention can include a cell line complete with heterologous glucose transport expression capability, or another membrane system with glucose transporter isoforms, and a detectable glucose compound, such as glucose or a glucose analog. Further included are standards for insulin stimulation, and protease inhibitors for standardizing the inhibition assays.
The invention further provides a method for the rational design of new antiretroviral treatments. The method provides that a candidate drug or lead compound be subjected, in the early stages of the drug development, to assays to measure effects on glucose transport. The assay method comprises the steps of identifying a therapeutic test compound; testing the compound by: determining that it inhibits a retroviral aspartyl protease; using the compound as a protease inhibitor in the screening method described above; assessing the compound for inhibition of glucose transport; and selecting those compounds which inhibit the aspartyl protease and which do not inhibit glucose transport. Candidates of greatest interest for further development are those which maximally inhibit the retroviral protease but do not substantially alter the glucose transport activity in either the presence or absence of insulin stimulation.
Further provided in the invention are methods of cell-free methods of rapidly screening protease inhibitors for specific molecular interactions with glucose transporters. Such specific molecular interactions are an indication of potential inhibition or undesirable side-effects of protease inhibitors. These methods provide for labeled glucose transporters and or labeled protease inhibitors. The invention provides for measuring the specific molecular interactions between either a membrane-associated or solubilized glucose transporter and a protease inhibitor.
Also provided in accordance with the present invention is a method of screening factors, compounds or conditions which alter reversible insulin-resistant glucose transport. Compounds identified by such a method would be excellent candidates for treating disease conditions comprising insulin resistance. The method comprises the steps of: providing a cell line producing one or more glucose transporters; incubating the cells with a detectable glucose or glucose analog in the presence of an inhibitor known to specifically inhibit the glucose transporter isoform of interest; including a compound or condition whose effects on reversing the inhibition are to be tested; and quantifying the reversal of glucose transport inhibition. Also provided are such assays in either the presence or absence of insulin or other glucose transport-altering substances.
Other features and advantages of the present invention will be understood by reference to the detailed description of the invention and examples that follow.